1. Field of the Invention
The present invention relates to the field of antibacterial therapy, and more specifically, methods and compositions for treating infections caused by Gram-negative bacteria in humans, animals and plants.
2. Description of the Related Art
Peptides with the ability to destroy bacteria are described in the prior art (C B Park, H S Kim, S C Kim. Biochem Biophys Res Commun. Mar. 6, 1998; 244(1): 253-7). Likewise, peptides capable of reacting with aminoglycans and transporting molecules of interest in eukaryotic or prokaryotic cells are disclosed in international patent application PCT No. WO 01/64738.
The amount of antibiotic molecules penetrating the bacterium depends on their structure and the mechanisms involved in transporting substrates. Gram-negative bacteria differ structurally from Gram-positive bacteria due the presence of two membranes that constitute the bacterial envelope. If all the bacteria have an internal membrane, then Gram-negative bacteria have an additional single external membrane. This external hydrophobic membrane forms a semi-permeable, barrier preventing antibiotics from penetrating, while porines, i.e., proteins forming channels, allow small hydrophilic solutes, such as nutritional elements and antibiotics of the penicillin and tetracycline family to enter, while preventing the penetration of large molecules and antibiotics of the macrolides/ketolides family.
An important reason for therapeutic failures to control the Gram-negative bacteria is the emergence of resistant strains. Certain resistances are related to a reduction of the permeability of bacterial membranes (quantitative/qualitative modifications of porines). Other resistances are due to the presence of a membranous protein causing the rejection of the antibiotic through an active efflux mechanism. The development of new types of antibacterial molecules or the application of non-active commercial antibiotics on the Gram-negative bacteria requires their entrance and delivery across selective bacterial membranes.
The object of the present invention is precisely to provide new methods and compositions that allow for effective treatment of infections caused by Gram-negative bacteria, even when these have developed resistance to antibiotics.
This object is obtained by using peptides capable of passing through the external membrane of Gram-negative bacteria, and through this membranal translocation, deliver molecules of interest that otherwise would not penetrate into the interior of bacteria, because of their physiochemical properties.
Penetration into the interior of the bacteria means that the peptides of the invention facilitate or allow the penetration of the molecules of interest into bacteria. The terms penetration and internalization are henceforth used synonymously.
The work performed in connection with the present invention concerned Bodipy and tetramethylrhodamine that are hydrophobic fluorescent molecules excluded by the external membrane of the Gram-negative bacteria. These fluorescent tracers were chosen in order to evaluate the internalization properties of the peptides of the invention being chemically bound to hydrophobic molecules. The translocation of fluorescent tracers in the Gram-negative bacterium was evaluated qualitatively on Escherichia coli and Pseudomonas aeruginosa. 